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Porcine SAA is not likely an apolipoprotein bound to circulating HDL3

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HAL Id: hal-01607287

https://hal.archives-ouvertes.fr/hal-01607287

Submitted on 6 Jun 2020

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Porcine SAA is not likely an apolipoprotein bound to circulating HDL3

Soler Laura, Ana Gutiérrez, P. David Eckersall, Nicola Merola, José J. Cerón, Théo Niewold

To cite this version:

Soler Laura, Ana Gutiérrez, P. David Eckersall, Nicola Merola, José J. Cerón, et al.. Porcine SAA is not likely an apolipoprotein bound to circulating HDL3. 8. European Colloquium on Acute Phase Proteins, Aug 2010, Helsinki, Finland. �hal-01607287�

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Porcine Serum Amyloid A is not 

likely an apolipoprotein bound to 

circulating HDL

Laura Soler, Ana Gutiérrez, P. David Eckersall, Nicola 

Merola, Jose J Cerón, Theo Niewold

Facultad de Veterinaria Departamento de Medicina y Cirugía  Animal

(3)

Veterinary clinical analysis research group, University of Murcia

Theo Niewold

(4)

SERUM AMYLOID A (SAA)

Acute phase, 14 kDa protein, multimers

SAA1 and SAA2

Mainly produced in liver

Circulating

Apolipoproteins

Neutral IEP

SAA3

Mainly produced locally

Not circulating

Alkaline IEP

       Introduction

(5)

 PORCINE SAA: 

THE BIG UNKNOWN

 Purification of pig SAA not published

 Nucleotide and amino acid sequences of each SAA 

isoform has not been reported

 No reports on identification by proteomic techniques

 Importance: swine resistance to amyloidosis 

development

       Introduction

(6)

OBJECTIVES

 Purifying SAA from porcine serum using traditional 

protocols and study its properties

 Analyzing predicted properties from sequences 

deposited in UniProtKB database by using ExPASy 

server sequence analysis tools

(7)

       Material and  Methods

3

­ Gradient density ultracentrifugation using 

iodixanol

1

­ Guanidine hydrochloride­eluted HIC

2

­ Ethanol­eluted HIC

SAA purification from pig serum

(8)

1. Rabbit anti­bovine SAA3 pAb Dr. Molenaar

2. Rat anti­porcine recombinant SAA mAb

Search UniProtKB database for porcine SAA sequences

and analyze with ExPASy tools (ProtParam, GRAVY)

       Material and  Methods

SDS­PAGE and immunoblotting analysis

Sequence analysis

(9)

 Hydrophobic interaction 

chromatography

70 kDa 65 kDa

Guanidine hydrochloride

Ethanol

        Results

(10)

HIC G FG G HIC Et 60 kDa SAA3b 30 kDa 20 kDa 15 kDa FG G HIC Et H IC G F G G H IC E t S A A 3 b M K

Rat mAb

Rabbit 

pAb

 Immunobloting analysis

SDS­PAGE

SAAp MK         Results

(11)

­ Porcine SAA binds to the HIC columns and 

need chaotropic agents, like guanidine 

hydrochloride, to elute.

­ The purification showed has a very low 

throughput and yield

­ It seems that the bulk of the SAA remain 

unbound to the HIC columns and is lost during the 

process

Results from HIC chromatography:

Results from HIC chromatography:

(12)

 Gradient Density Ultracentrifugation

Sudan

Coomassie

VLDL

LDL

HDL

Proteins

        Results

(13)

Rabbit pAb

60 kDa SAA3b SAAp MK HDL HDL

HDL

Proteins

Rat mAb

 Immunobloting analysis

        Results 40 kDa 28 kDa 14 kDa

(14)

Results from SAA­HDL isolation by gradient 

Results from SAA­HDL isolation by gradient 

density ultracentrifugation:

density ultracentrifugation:

­ Porcine SAA is present in HDL­rich fractions

­ Immunoblot analysis indicates that the bulk of 

SAA in porcine serum is not bound to HDL, but 

soluble and present in the serum protein layer

(15)

MKLSTGIIFCFLILGVSSQRWASFLKEAGQGAKDMW RAYSDMREANYKNSDKYFHARGNYDAAQRGPGGA WAAKVISDARENVQRVTDLFKHGDSGHGVEDSRAD QAANAWGRSGKDPNHFRPRGLPDKY Q2HXZ9 B9P414

 

Q2HXZ9

 Chang et al., 2007, 

Liver

, 130 aa, translated from 

mRNA

 

B9P414

 Rodriguez et al., 2007, 

Mammary gland

, 89 aa, translated 

from mRNA

        Results

(16)

Q2HXZ9

 

Molecular weight: 14511.1

Theoretical pI: 9.48

Grand average of hydropathicity (GRAVY): ­0.86

B9P414

Molecular weight: 9854.6

Theoretical pI: 9.15

Grand average of hydropathicity (GRAVY): ­1.254

        Results

 Sequence analysis

(17)

Results from sequence analysis:

Results from sequence analysis:

SAA3­like sequences:

­Low hidrophobicity

­High isoelectric point

­SFLK motif

Protein purification vs. sequence analysis:

Protein purification vs. sequence analysis:

Theoretically low hydrophobic protein that can 

behave as hydrophobic, but the bulk of the 

protein is found to be hydrophilic

(18)

Molenaar et al. Serum Amyloid A3 from Bovine Mammary Tissue; 

Progress Toward its Function and Purification. 7th International 

Acute Phase Protein Congress Barcelona. 2nd October 2008

Teorethical properties

Teorethical properties

MW – 12.7 Kd monomer

Isoelectric point – 

pI 9.56­9.59

  monomer

Average 

low hydrophobicity

Evidenced properties

Evidenced properties

­

 Behaves as if it were highly hydrophobic – binds 

strongly to hydrophobic columns

­

 When purified from milk, the bulk of SAA was found 

in skimmed milk, not in cream. HIC not successful to 

(19)

The results obtained in this study suggest 

that circulating SAA in the pig might be a 

SAA3­like protein, which would preclude its 

function as an apolipoprotein and would 

explain the high resistance of pigs to the 

development of AA­amyloidosis

CONCLUSION

(20)

Thank you

Kiitoksia

Gracias

Tack så mycket

Mange tak

Vielen Dank

Dank u wel

Merci

Grazie mille

Obrigado

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